Novel Bi-Modular GH19 Chitinase with Broad pH Stability from a Fibrolytic Intestinal Symbiont of Eisenia fetida, Cellulosimicrobium funkei HY-13.

Lu Bai,Dong-Ha Shin,Chung-Wook Chung,Jonghoon Kim,Kwang-Hee Son,Do Kim,Bon-Hwan Ku,Ho-Yong Park

doi:10.3390/biom11111735

Abstract

Endo-type chitinase is the principal enzyme involved in the breakdown of N-acetyl-d-glucosamine-based oligomeric and polymeric materials through hydrolysis. The gene (966-bp) encoding a novel endo-type chitinase (ChiJ), which is comprised of an N-terminal chitin-binding domain type 3 and a C-terminal catalytic glycoside hydrolase family 19 domain, was identified from a fibrolytic intestinal symbiont of the earthworm Eisenia fetida, Cellulosimicrobium funkei HY-13. The highest endochitinase activity of the recombinant enzyme (rChiJ: 30.0 kDa) toward colloidal shrimp shell chitin was found at pH 5.5 and 55 °C and was considerably stable in a wide pH range (3.5–11.0). The enzyme exhibited the highest biocatalytic activity (338.8 U/mg) toward ethylene glycol chitin, preferentially degrading chitin polymers in the following order: ethylene glycol chitin > colloidal shrimp shell chitin > colloidal crab shell chitin. The enzymatic hydrolysis of N-acetyl-β-d-chitooligosaccharides with a degree of polymerization from two to six and colloidal shrimp shell chitin yielded primarily N,N′-diacetyl-β-d-chitobiose together with a small amount of N-acetyl-d-glucosamine. The high chitin-degrading ability of inverting rChiJ with broad pH stability suggests that it can be exploited as a suitable biocatalyst for the preparation of N,N′-diacetyl-β-d-chitobiose, which has been shown to alleviate metabolic dysfunction associated with type 2 diabetes.

Highlights

Chitin is a renewable biopolymer that is extensively distributed in various ecosystems as the second most abundant structural polysaccharide after β-1,4-D-glucan
The 966-bp ChiJ gene (GenBank accession number: OK482705) coding for an extracellular GH19 chitinase was identified from the whole genome sequence of C. funkei HY-13
SMART, and protein BLAST analyses of the primary structure of premature ChiJ displayed that it might be a bi-modular chitinase consisting of a chitin-binding domain type 3 (ChtBD3: from Ala52 to Trp94) in the N-terminus region and a putative catalytic GH19 domain in the C-terminus region (Figure 1)

Summary

Introduction

Chitin is a renewable biopolymer that is extensively distributed in various ecosystems as the second most abundant structural polysaccharide after β-1,4-D-glucan This insoluble polysaccharide, which is comprised of N-acetyl-D-glucosamine (GlcNAc) repeating units combined by β-1,4-D-glycosidic linkages, is present in the cell walls of fungi and the exoskeletons of insects and crustaceans, including shrimp, krill, and crabs [1,2]. The biological recycling of chitin polysaccharides is predominantly carried out by a variety of indigenous chitinolytic bacterial and fungal species [3,4]. They generally produce endo-type chitinases (EC 3.2.1.14) and exo-type chitinases (EC 3.2.1.52), such as chitobiosidases and N-acetyl-β-D-glucosaminidases, for the cooperative deconstruction of chitins to low molecular weight substances including N-acetyl-β-D-chitooligosaccharides (N-acetyl-β-D-COSs) and GlcNAc [2,5]. Endo-type chitinases are essential enzymes responsible for the degradation of chitins and, have attracted much attention as potential candidates for biocontrol of fungal pathogens, the 4.0/).

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Conclusion